The invention relates to a method and an apparatus for detecting fluorophore labelled substances in an electrophoretic separation medium.
When detecting fluorophore labelled substances in an electrophoretic separation medium, e.g. in connection with DNA sequencing, it is known that upon excitation, at some stage, the signal generated by a detector in response to the detected intensity of the fluorescence emitted by the substances, may fade out, e.g. due to the fact that there is too little fluorophore labelled DNA in the sample. There may also be other reasons for the signal to fade out.
It is also known that in some applications, e.g. fragment analysis, the opposite situation may arise, namely that the intensity of the fluorescence will be too high for the detector to handle, i.e. the detector will become saturated.
In both these cases, information about the separated substances may be lost. This can e.g. lead to that it will be impossible to determine the DNA sequence.
Proteins and carbohydrates are two other examples of substances that may be labelled with fluorophores and separated in an electrophoretic separation medium.
Patent abstracts of Japan, abstract of JP-A-7-151687, describes the prevention of reading errors of a basic sequence in a fluorescence detection type electrophoretic apparatus by monitoring the intensity of the excitation light. An alarm is generated when the detection signal from a photodetector is smaller than a predetermined reference value because of an abnormal intensity of the excitation light.
The object of the invention is to eliminate the problems mentioned above.
This is attained by the method according to the invention by illuminating the fluorophore labelled substances, detecting fluorescence emitted by said fluorophore labelled substances upon illumination, and varying the illumination of the fluorophore labelled substances to vary the intensity of the fluorescence.
The above object is also attained by the apparatus according to the invention in that illuminating means are provided for illuminating the fluorophore labelled substances, detecting means are provided for detecting fluorescence emitted by said fluorophore labelled substances upon illumination, and illumination varying means are provided for varying the illumination of the fluorophore labelled substances to vary the intensity of the fluorescence.